Establishment of a multiplex real time quantitative PCR method for CMV promoter nucleic acid sequences detection
10.3867/j.issn.1000-3002.2014.02.026
- VernacularTitle:巨细胞病毒启动子序列检测的复合实时定量 PCR 方法的建立
- Author:
Yufa MIAO
;
Sanlong WANG
;
Xiaobing ZHOU
;
Yan HUO
;
Xingchao GENG
;
Jianjun LYU
;
Jufeng WANG
;
Bo LI
- Publication Type:Journal Article
- Keywords:
real time quantitative PCR;
CMV promoter;
β-actin gene
- From:
Chinese Journal of Pharmacology and Toxicology
2014;(2):296-301
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVE To establish and validate a multiplex real time quantitative PCR method for cyto megalovirus(CMV)pro moter nucleic acid sequence detection.METHODS Probes and primers were designed according to CMV pro moter sequence and mouse β-actin house-keeping gene,the a mpli-fication specificity was analyzed using SYBR Green I dissociation curve.The reaction syste m was opti-mized,the sensitivity,linearity and reproducibility of the method were validated.RESULTS Forward primer sequence for CMV pro moter sequence were 5′AGACTTGGAAATCCCCGTGAGT3′;reverse prim-er sequence were 5′CGTATTAGTCATCGCTATTACCATGGT3′;probe sequence were 5′AACCGC-TATCCACGCCCATTGATG3′. Forward primer sequence for β-actin gene were 5′CCTGAG-GCTCTTTTCCAGCC3′; reverse primer sequence were 5′TAGAGGTCTTTACGGATGTCAACGT3′;probe sequences were 5′TCCTTCTTGGGTATGGAATCCTGTGGC3′.Reaction efficiency of the CMV standard curve reached 100%, correlation coefficient reached 0.9978, quantification margin was between 1 .5 ×102 and 1 .5 ×107 copies,and sensitivity of the reaction reached 30 copies.CONCLUSION The multiplex method that could absolutely quantify the copies of CMV pro moter sequence is established.
